title: Entry of a cationic lytic-type peptide into the cytoplasm via endocytosis-dependent and -independent pathways in human glioma U251 cells.
abstract: Cationic lytic-type peptides have been studied for clinical application in various infections and cancers. This study aimed to determine the functions of our specially designed lytic peptide. To investigate the functional mechanism at the cell membrane level, we used giant unilayer vesicles (GUVs) mimicking cell membranes. In GUVs treated with FITC-labeled lytic peptide (lytic-FITC), fluorescence increased in a time-dependent manner. However, no inner fluorescence was detected in GUVs treated with lytic peptide and calcein. Next, distribution of lytic-FITC peptide on the cell membrane and in the cytoplasm was examined in a living human glioma U251 cell line. In the immunocytochemical study, some lytic peptide stains colocalized with early endosome antigen protein 1 (EEA-1). In cells treated with lytic peptide, the immunofluorescence intensity of lytic peptide increased in a concentration and treatment time-dependent manner. Cytotoxic activity of lytic peptide decreased after pretreatment with the endocytosis inhibitors cytochalasin D, chlorpromazine and amiloride. These findings suggest that lytic peptide exerts cytotoxic activity after cellular uptake via an endocytosis pathway. In conclusion, the influx mechanism of lytic peptide was shown to include not only disintegration and pore formation at the cell membrane, but also cell entry via endocytosis dependent and independent pathways.
authors.affiliation: Department of Pharmacoepidemiology, Graduate School of Medicine and Public Health, Kyoto University Yoshidakonoecho, Sakyoku, Kyoto city, Kyoto 606-8501, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Ohara
authors.forename: Masayuki
authors.initials: M
authors.lastname: Kohno
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Hamada
authors.forename: Koji
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D000584:Amiloride / Q000494:pharmacology; D000595:Amino Acid Sequence; D001692:Biological Transport; D002412:Cations; D045744:Cell Line, Tumor; D002746:Chlorpromazine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003603:Cytotoxins / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D013194:Staining and Labeling; D053835:Unilamellar Liposomes / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D002412:Cations; D003603:Cytotoxins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; D053835:Unilamellar Liposomes; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D015638:Cytochalasin D; D000584:Amiloride; D016650:Fluorescein-5-isothiocyanate; D002746:Chlorpromazine; C007740:fluorexon
keywords: Anti-cancer peptide; Cationic peptide; Endocytosis; Lytic-type peptide